The spider genus Hypochilus is a relictual lineage of Nearctic species distributed disjunctly across the United States in three montane regions (California, southern Rocky Mountains, southern Appalachia). Phylogenetic resolution of species relationships in Hypochilus has been challenging, with different data types recovering sometimes conflicting topologies. Furthermore, conserved morphology coupled with extreme genetic divergence and paraphyletic topologies at the species level has led to uncertain species limits in some complexes. Using a combination of ultraconserved elements (UCEs), mitochondrial CO1 by-catch, and studies of morphology, we have reconstructed Hypochilus interspecies relationships and have more critically evaluated the possibility of cryptic species in California and in southern Appalachia. Phylogenomic data from hundreds of nuclear loci strongly support the monophyly of regional clades, and furthermore support a ((California, Appalachia), southern Rocky Mountains)) topology. We hypothesize late Cenozoic age divergences in North America, but acknowledge that more research is needed to reliably date divergences in this genus. Within the southern Appalachian fauna, five species are resolved as four lineages (H. thorelli Marx, 1888 and H. coylei Platnick, 1987 are clearly sister taxa), but the interrelationships of these four lineages remain unresolved despite phylogenomic-scale data. The Appalachian species H. pococki Platnick, 1987 is recovered as monophyletic, which differs from prior mitochondrial evidence, but is clearly highly genetically structured at the nuclear level. Although algorithmic analyses of the nuclear data indicate many species (e.g., all populations as species), scanning electron microscopy (SEM) surveys of male morphology instead reveal striking morphological stasis, clearly illustrating the cryptic species problem. Within the California clade, nuclear and mitochondrial lineages of H. petrunkevitchi Gertsch, 1958 correspond directly to drainage basins of the southern Sierra Nevada, with H. bernardino Catley, 1994 nested within H. petrunkevitchi and sister to the southernmost basin populations. Again, algorithmic analyses of nuclear and mitochondrial data suggest many species, but we take a more conservative approach, allowing for population genetic structure within species. Combining nuclear, mitochondrial, geographic and morphological evidence we describe a new species from the Tule River and Cedar Creek drainages, Hypochilus xomote sp. nov. We also emphasize the conservation issues that face several microendemic, habitat-specialized species in this remarkable genus.

Sequence capture libraries were prepared using a UCE capture protocol for arachnids (Starrett et al. 2017, Hedin et al. 2019), with arachnid probes designed by Faircloth (2017). Sequencing was done at the Brigham Young University DNA Sequencing Center on an Illumina HiSeq 2500 150 cycle paired-end sequencing platform. Published data for two H. pococki specimens (H595 and H232, from Starrett et al. 2017), one H. kastoni Platnick, 1987 (G2519, Hedin et al. 2019), and Ectatosticta (Ramírez et al. 2021) were used from prior studies. Raw reads were filtered using the illumiprocessor wrapper (Faircloth 2013) within PHYLUCE v1.6 (Faircloth 2016), after which cleaned reads were assembled using Trinity v2.0.6 (Grabherr et al. 2011) and Velvet v1.0.19 (Zerbino and Birney 2008) on the HPC Cluster at UC Riverside. These assemblies were combined and resulting contigs were matched to probes with minimum identity and minimum coverage values of 80 (--min-identity 80 --min-coverage 80). UCE loci were aligned and trimmed within phyluce using MAFFT (Katoh and Standley 2013) and Gblocks (Castresana 2000; Talavera and Castresana 2007) with relatively liberal settings for GBLOCKS (--b1 0.5 --b2 0.5 --b3 10 --b4 8).